A major trend in the detergent industry is to provide cleaning formulations that are functional at low wash temperature, are stable in the presence of laundry additives, and can be formulated as liquid, dry or granular compositions, while sufficient washing or cleaning ability is retained. These formulations generally require the use of enzymes, particularly proteases to fulfill this need.
In order to be useful as a detergent enzyme, it is desirable for a protease to possess proteolytic activity against proteinaceous substances over a wide pH and temperature range; exhibit catalytic stability in the presence of surfactants, builders, oxidizing agents and other detergent components; and maintain activity during storage (adequate shelf-life).
The most widely used proteases in cleaning compositions are the alkaline proteases derived from various strains of Bacillus. Such proteases, which are marketed by Novo/Nordisk Copenhagen, Denmark under trade names such as Esperase, Alcalase, or subtilisin Carlsberg, and Savinase, derived from Bacillus licheniformis have desirable alkaline stability properties and proteolytic activities. These enzymes have been well characterized and are widely used by detergent manufacturers. While effective in hot water washing, the temperature optima of the Bacillus enzymes is between 60.degree.-70.degree. C., which is above the normal temperatures used for warm (30.degree.-40.degree. C.) and cool (15.degree.-30.degree. C.) water washings. Moreover, the Bacillus alkaline proteases have less than desirable stability to oxidizing agents, and are completely unstable in chlorine bleaches, which precludes their use with chlorine bleaches, automatic dishwasher detergents, etc.
Variants of subtilisin, a serine protease produced by Gram-positive bacteria, have been produced using both random and site-directed mutagenesis of the subtilisin gene to affect changes in catalytic efficiency (value of k.sub.cat /K.sub.M), substrate specificity and tertiary structure. Specific subtilisin variants have also been characterized which show improved stability to oxidation, increased protease activity or improved washing ability (U.S. Pat. No. 4,760,025 issued Jul. 26, 1988 to Estell, et al; WO89/06279 published Jul. 13, 1989 to Hastrup, et al; and WO89/09819 published Oct. 19, 1989 to Bryan, et al).
Streptomyces griseus, an organism used for the commercial production of the alkaline protease "Pronase" (available from Calbiochem), secretes many extracellular proteins (Jurasek, et al (1971) Can J Biochem 49:1195-1201). Protease A and protease B, two of the serine proteases secreted by S. griseus strain IMRU 3499, have sequences which are 61% homologous on the basis of amino acid identity and 67% homologous in the coding region based on DNA identity (Henderson, et al (1987) J Bacteriol 169(8):3778-3784). Japanese Patent No. 977824 titled "A Method of Manufacturing Alkaline Proteases" discloses the strain S. griseus var. alkaliphilus No. 33 (which is deposited with the Microbiology and Industrial Institute Microorganism Deposit No. 2086) and a method of obtaining a crude extract of an enzyme having an activity optimum at greater than pH 12 and proteolytic activity in 0.2 moles of sodium hydroxide solution.
Recently, a partial amino acid sequence of a third S. griseus protease, designated S. griseus alkaline protease (SGP), was reported in Tsujibo, et al (1990) Agric Biol Chem 54(8): 2177-2179. Based on a sequence comparison of the partial SGP sequence with the protease of the present invention, substantial amino acid differences are noted: Arg.sub.15 .fwdarw.Asp; Arg.sub.23 .fwdarw.Ser; and Ser.sub.26 .fwdarw.Ala, all numbered in accordance with SEQ ID No. 1 herein. No further characterization of this enzyme has been reported.
Despite the existence of a multitude of known proteases and the application of recombinant DNA technology to the study and production of protease analogs, the art has yet to develop proteases completely satisfactory for use in modern cleaning formulations.